Caveolae and L-type Calcium Channels in Cardiomyocytes

描述（由申请人提供）：项目概述：心肌细胞表面膜中通过L型Ca通道的Ca内流调节多种细胞过程，包括收缩、分泌、细胞信号传导和基因表达。本研究的长期目标是：1）基于相关蛋白的独特大分子复合物来区分心肌细胞中不同的L型Ca通道群体; 2）确定这些不同的通道群体如何特异性地促进各种Ca调节的细胞过程; 3）了解不同Ca通道群体的改变如何促进心血管疾病。这些目标将首先集中在Cav1.2 L型钙通道定位到专门的膜微区称为小窝，这是由签名蛋白小窝蛋白-3（Cav-3）定义。将定义Cav1.2通道对与最近发现的Cav-3突变相关的长QT综合征发生的贡献。本研究利用免疫沉淀技术、GST-Cav-3 pulldown技术、免疫共聚焦显微镜、免疫金电镜、siRNA敲除靶蛋白、Cav1.2通道及其关键相关蛋白的异源表达以及条件性敲除小鼠Cav-3等技术，主要研究三个方面的内容：1）明确心室肌细胞膜小窝Cav1.2通道的相对丰度和组成; 2）确定Cav-3和相关的核心支架蛋白是否是心室肌细胞中正常的基础L型Ca电流及其β-AR调节所必需的; 3）确定长QT相关的Cav-3基因突变对Cav 1.2 L型Ca通道的影响。 相关性：这些研究将提供定位于小窝的Ca通道蛋白亚群的分子定义，从而提供与心脏小窝的特征蛋白小窝蛋白-3突变相关的长QT综合征和危及生命的心律失常的发生机制的见解。此外，这项研究将影响我们对心力衰竭和房颤的理解，因为在这些疾病中已经检测到了小窝和钙通道的改变。最终，对小窝和L型Ca 2+通道的分子理解有可能激发这些流行心血管疾病的新疗法。

DESCRIPTION (provided by applicant): Project Summary: The influx of Ca through L-type Ca channels in surface membrane of cardiomyocytes regulates a variety of cellular processes including contraction, secretion, cell signaling, and gene expression. The long-term goals of this research are 1) to discriminate between distinct populations of L-type Ca channels in cardiomyocytes based on the unique macromolecular complex of associated proteins; 2) to determine how these different populations of channels specifically contribute to various Ca-regulated cellular processes; and 3) to understand how alterations in the different populations of Ca channels contribute to cardiovascular disease. These goals will be approached initially focusing on Cav1.2 L-type Ca channels localized to the specialized membrane microdomains known as caveolae which are defined by the signature protein caveolin-3 (Cav-3). The contribution of Cav1.2 channels to the genesis of the long QT syndrome associated with recently identified mutations in Cav-3 will be defined. Using immunoprecipitation techniques, GST-Cav-3 pull-down, immunoconfocal microscopy, immunogold electron microscopy, siRNA knockdown of targeted proteins, heterologous expression of Cav1.2 channels and key associate proteins, and conditional knockout of Cav-3 in mice, we will address three specific aims: 1) Define the relative abundance and composition of the Cav1.2 channels localized to caveolae in ventricular myocytes; 2) Determine if Cav-3 and associated core scaffolding proteins are essential for normal basal L-type Ca current and its (¿-AR regulation in ventricular myocytes; 3) Determine the impact of long QT-related Cav-3 genetic mutations on Cav1.2 L- type Ca channels. Relevance: These studies will provide molecular definition of the subpopulation of Ca channel proteins localized to caveolae and thus offer mechanistic insights into the genesis of the long QT syndrome and life-threatening arrhythmias associated with mutations in the signature protein of caveolae in the heart, caveolin-3. Furthermore, this research will impact our understanding of heart failure and atrial fibrillation given that alterations in caveolae and Ca channels have been detected in these diseases. Ultimately, a molecular understanding of caveolae and L-type Ca2+ channels has the potential to inspire new therapies for these prevalent cardiovascular diseases.